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Cholera is a leading global public health threat, especially in developing countries. This study aimed to determine the changing determinants of cholera related to water-sanitation practices between 1994-1998 and 2014-2018 in Dhaka, Bangladesh. Data of all cause diarrhea cases were extracted from the Diarrheal Disease Surveillance System of the International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, and analysis was performed among three groups: Vibrio cholerae detected as sole pathogen, V. cholerae detected as mixed infection, and detection of no common enteropathogen in stool specimens (reference). Using sanitary toilet, drinking tap water, drinking boiled water, family size greater than five, and slum dwelling were the main exposures. Overall, 3,380 (20.30%) and 1,290 (9.69%) patients were positive for V. cholerae during 1994-1998 and 2014-2018, respectively. In 1994-1998, use of sanitary toilet (adjusted odds ratio [aOR]: 0.86, 95% CI: 0.76-0.97) and drinking tap water (aOR: 0.81, 95% CI: 0.72-0.92) were found to be negatively associated and in 2014-2018, drinking tap water (aOR: 1.47, 95% CI: 1.21-1.78) and slum dwelling (aOR: 1.43, 95% CI: 1.10-1.86) were found to be positively associated with V. cholerae infection after adjusting for age, sex, monthly income, and seasonality. Because the determinants of cholera such as drinking tap water can change over time in developing cities, ameliorating the water, sanitation, and hygiene (WASH) situation is of paramount importance. In addition, in settings such as urban slums, where long-term WASH monitoring might be difficult to achieve, mass vaccination with oral cholera vaccine should be introduced to control cholera.

Background: Information on comparative clinical and host characteristics of under-2 children with watery diarrhea caused by rotavirus, Enterotoxigenic Escherichia coli (ETEC), and Vibrio cholerae as single pathogens is lacking. We sought to investigate the sociodemographic, clinical, and host characteristics of under-2 children hospitalized due to these pathogens., Methodology: We conducted a hospital-based case-control study using the icddr,b Diarrheal Diseases Surveillance System. Children of either sex, <2 years with diarrhea, who attended the hospital during 2014 to 2018, constituted the study population. Stool specimens having a single pathogen like rotavirus, ETEC, or Vibrio cholerae constituted the cases and stool specimens having no detectable common enteropathogens comprised the controls. Multinomial logistic regression analysis was done where control was the reference group., Results: A total of 14 889 patients were enrolled, 6939 of whom were under-2 children, and 5245 (76%) constituted our study population. Among them 48% (n = 2532), 3% (n = 148) and 1% (n = 49) had rotavirus, ETEC, and Vibrio cholera , respectively. A control group (diarrhea without these 3 or Shigella , Salmonella , Aeromonas ) accounted for 48% (n = 2516). In multinomial regression model, children with rotavirus (adjusted odds ratio [aOR], 1.36; 95% confidence interval [95% CI], 1.19-1.55) less often presented with dehydrating diarrhea compared to those with ETEC (aOR, 1.54; 95% CI, 1.05-2.26) and cholera (aOR, 2.25; 95% CI, 1.11-4.57). Rotavirus diarrhea was associated (aOR, 1.25; 95% CI, 1.07-1.46) with those who received antimicrobials prior to hospital admission and protectively associated with drinking tap water (aOR, 0.84; 95% CI, 0.73-0.95); however, ETEC diarrhea had protective association (aOR, 0.62; 95% CI, 0.43-0.92) with children who received antimicrobials prior to hospital admission and was associated with drinking tap water (aOR, 1.78; 95% CI, 1.19-2.66). Use of intravenous fluid was associated with cholera (aOR, 10.36; 95% CI, 4.85-22.16) and had protective association with rotavirus episodes (aOR, 0.64; 95% CI, 0.45-0.91)., Conclusions: Clinical presentations and host characteristics of rotavirus, ETEC, and Vibrio cholerae diarrhea differed from each other and the information may be helpful for clinicians for better understanding and proper management of these children.

A new gene, mutK, of Vibrio cholerae, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the mutK gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of Escherichia coli mutS, mutL, mutU, and dam mutants. The spontaneous mutation frequency of a chromosomal mutK knockout mutant was almost identical to that of wild-type V. cholerae cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the mutK gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the mutK gene product. The mutK gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of mutK, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the E. coli vsr gene. ORF1, located farther upstream of mutK, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of mutK, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The mutK gene has recently been positioned in the ordered cloned DNA map of the genome of the V. cholerae strain from which the gene was isolated (10).

The unprecedented genesis of a novel non-O1 Vibrio cholerae strain belonging to serogroup O139, which caused an epidemic in late 1992 in the Indian subcontinent, and its subsequent displacement by El Tor O1 vibrios after 18 months initiated a renewed investigation of the aspects of the organism that are related to pathogenesis. The reappearance of V. cholerae O139 with altered antibiotic sensitivity compared to O139 Bengal (O139B) in late 1996 has complicated the epidemiological scenario of V. cholerae and has necessitated an examination of possible rearrangements in the genome underlying such rapid changes in the phenotypic traits. With a view to investigating whether the phenotypic changes that have occurred are associated with alteration in the genome, the genome of the resurgent V. cholerae O139 (O139R) strains were examined. Pulsed-field gel electrophoresis analysis of NotI- and SfiI-digested genomic DNA of O139R isolates showed restriction fragment length polymorphism including in the cholera toxin (CTX) genetic element locus and with O139B isolates. Analyses of the organization of the CTX genetic elements in O139R strains showed that in contrast to two copies of the elements connected by two direct-repeat sequences (RS) in most of the genomes of O139B isolates, the genomes of all O139R strains examined, except strain AS192, have three such elements connected by a single RS. While the RS present in the upstream of the CTX genetic elements in the genome of O139R is of O139B origin, the RS connecting the cores of the elements has several new restriction sites and has lost the BglII site which is supposed to be conserved in all O1 strains and O139B. The endonuclease I-CeuI, which has sites only in the rrn operons in the genomes of all organisms examined so far, has 10 sites in the genomes of O139R strains, compared to 9 in the genomes of O139B strains. The recent isolates of V. cholerae O139 have thus gained one rrn operon. This variation in the number of rrn operons within a serogroup has not been reported for any other organism. The results presented in this report suggest that like the pathogenic El Tor O1 strains, the genomes of O139 strains are undergoing rapid alterations.

A combined physical and genetic map of the genome of strain SG24 of Vibrio cholerae O139 Bengal, a novel non-O1 strain having epidemic potential, has been constructed by using the enzymes NotI, SfiI, and CeuI. The genome of SG24 is circular, and the genome size is about 3. 57 Mb. The linkages between 47 NotI and 32 SfiI fragments of V. cholerae SG24 genomic DNA were determined by combining two approaches: (i) identification of fragments produced by enzyme I in fragments produced by enzyme II by the method of fragment excision, redigestion, and end labeling and (ii) use of the linking clone libraries generated from the genome of classical O1 strain 569B. The linkages between nine CeuI fragments were determined primarily by analyses of partial fragments of the CeuI-digested genome. More than 80 cloned homologous and heterologous genes, including several operons, have been positioned on the physical map. The map of the SG24 genome represents the second map of a V. cholerae genome, and a comparison of this map with that of classical O1 strain 569B revealed considerable diversity in DNA restriction sites and allowed identification of hypervariable regions. Several genetic markers, including virulence determinant genes, are in different positions in the SG24 and 569B genomes.

By using a low-resolution macrorestriction map as the foundation (R. Majumder et al., J. Bacteriol. 176:1105-1112, 1996), an ordered cloned DNA map of the 3.2-Mb chromosome of the hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. A cosmid library the size of about 4,000 clones containing more than 120 Mb of V. cholerae genomic DNA (40-genome equivalent) was generated. By combining landmark analysis and chromosome walking, the cosmid clones were assembled into 13 contigs covering about 90% of the V. cholerae genome. A total of 92 cosmid clones were assigned to the genome and to regions defined by NotI, SfiI, and CeuI macrorestriction maps. Twenty-seven cloned genes, 9 rrn operons, and 10 copies of a repetitive DNA sequence (IS1004) have been positioned on the ordered cloned DNA map.

The secY gene of Vibrio cholerae has been cloned and the complete nt sequence determined. It codes for a protein of 438 aa residues which functions as a translocator through which proteins cross the inner membrane. It can substitute for the Escherichia coli SecY protein and can suppress the phenotypic traits associated with E. coli secY mutants. The V. cholerae secY gene has about 71 and 83% similarity at the nt and aa levels respectively with the E. coli secY gene. Vibrio cholerae secY, similarly to the E. coli secY, is flanked by the genes encoding the ribosomal large subunit proteins L15 and L36. When expressed from the lac promoter, V. cholerae secY partially complements E. coli secY mutation even in absence of IPTG, while the E. coli secY gene complements only when IPTG is present. Presence of multiple SD sequences and a putative downstream (DS) box imply that the V. cholerae secY gene might have high translational efficiency. A V. cholerae mutant unable to translocate CTB through the inner membrane has been isolated. The secretion deficient phenotype of the mutant can be reversed by introducing the cloned V. cholerae secY gene.

The intron-encoded enzyme I-CeuI provides an excellent tool for rapidly examining the organization of genomes of related species of bacteria. Vibrio cholerae strains belonging to serovars O1 and O139 have 9 I-ceuI sites in their genomes, and V. cholerae strains belonging to serovars non-O1 and non-O139 have 10 I-ceuI sites in their genomes. This information can be used as a criterion to differentiate O1 strains from non-O1 and non-O139 strains. To our knowledge, intraspecies variation in the number of rrn operons has not been reported in any other organism. Our data revealed extensive restriction fragment length polymorphism based on a comparison of the I-ceuI digestion profiles of strains belonging to different serovars and biovars. From the analysis of partial digestion products, I-CeuI macrorestriction maps of several classical, El Tor, and O139 strains were constructed. While the linkage maps are conserved within biovars, linkage maps vary substantially between biovars.

The Vibrio cholerae rpoH gene coding for the heat-shock sigma factor, sigma 32, has been cloned and shown to functionally complement Escherichia coli rpoH mutants. The nt sequence of the gene has been determined and the deduced aa sequence is more than 80% homologous to the E. coli rpoH gene product. Downstream of the V. cholerae rpoH gene, an unidentified dehydrogenase gene (udhA) is present on the opposite strand facing rpoH. The predicted secondary structure of the 5'-proximal region of V. cholerae rpoH mRNA is apparently different from the conserved secondary structures of the rpoH mRNA reported for several bacterial species. The 'RpoH box', a stretch of 9 aa (QRKLFFNLR) unique to sigma 32 factors, and the 'downstream box' sequence complementary to a part of the 16S rRNA, have been detected.

A combined physical and genetic map of the genome of the classical O1 hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. The enzymes NotI, SfiI and CeuI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis. The digests produced 37, 22, and 7 fragments, respectively. The CeuI maps of the genomes of strains 569B and O395, constructed by partial restriction digestion, were identical, and the data are consistent with the concept of circular chromosomes. The genome size of each of the strains was estimated to be about 3.2 Mb. The NotI and SfiI digestion profiles of the genomic DNAs of strains 569B and O395 exhibited distinct restriction fragment length polymorphism. The linkages between the 37 NotI fragments of the genome of strain 569B were determined by combining three approaches: isolation of linking clones, analysis of partial digestion fragments, and identification of NotI fragments in isolated CeuI and SfiI fragments. To align linked fragments precisely, NotI-digested genomic DNA was end labeled and separated in the same gel with the NotI-digested DNA to be probed with linking clones. This also allowed the identification of smaller restriction fragments that are not visible in ethidium bromide-stained gels. The presence of repetitive DNA sequences in the V. cholerae 569B genome has been demonstrated. Twenty cloned homologous and heterologous genes and seven rrn operons have been positioned on the physical map. The two copies of the Ctx genetic element in the genome of strain 569B are located about 1,000 kb apart.

Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity. The synthesis of all the three proteins is regulated by the osmolarity of the growth medium. The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded. The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units. Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit. Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein. The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium. The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that of OmpU. Southern blot hybridization of V. cholerae genomic DNA digested with several restriction endonucleases with nick-translated E. coli ompF as the probe revealed no nucleotide sequence homology between the ompU and ompF genes. OmpU is also not antigenically related to OmpF. Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V. cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium. OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the possible virulence determinants in V. cholerae (V. L. Miller and J. J. Mekalanos, J. Bacteriol. 170:2575-2583, 1988).

PFGE analysis of the NotI- and SfiI-digested genome of Vibrio cholerae O139 strains isolated from different epidemic regions of India showed that all the strains are of clonal origin and the genome size is about 2.2 Mb. An analysis of the electrophoretic profiles of the genome of O139 strains, the RFLP of the cholera toxin (ctx) gene and Southern blot hybridization of NotI-digested genomes of classical, El Tor and O139 with a NotI-linking clone of classical strain 569B, suggest that these strains closely resemble V. cholerae O1 biotype El Tor, but are widely different from the classical O1 vibrios. Using restriction enzymes which cleave a single site in either the core region or in the direct repeat sequence (RS) of the CTX genetic element, it has been shown that the genome of most of the O139 strains has two copies of the ctx gene in tandem connected by two RSs. The chromosomal location of the CTX genetic element in the O139 strain is the same as that reported for El Tor vibrios. The organization of the virulence gene cassettes in different O139 strains shows genetic heterogeneity in the population. Whilst most of the epidemic strains have two copies of the CTX genetic element, in some strains the number of elements has been amplified and in at least one strain a single copy of the element has been deleted.

A 25-kDa outer membrane protein, induced following treatment of Vibrio cholerae cells with beta-lactam antibiotics and constituting about 8-10% of the total outer membrane proteins of beta-lactam-resistant mutants, has been purified to homogeneity. It is a basic (pI 8.5) protein rich in beta-sheet structure and is a homodimer, the monomers being held together by hydrophobic interactions. The effective hydrophobicity of the protein is low, and a large part of the protein is exposed on the surface of the outer membrane. The protein does not have beta-lactamase or autolytic activity and is not a penicillin-binding protein. The Stoke's radius of the 25-kDa protein (26 A) is comparable to the pore size of the V. cholerae OmpF-like porin. Proteoliposome swelling assay showed that the 25-kDa protein might block the pores of OmpF through which beta-lactam antibiotics normally enter the cells. Twenty-two amino acid residues from the N-terminal end of the 25-kDa protein have been sequenced, and a 32-mer oligonucleotide probe was synthesized using the amino acid residues 2-12. This probe was used to identify the gene encoding the 25-kDa protein. The beta-lactam-resistant cells are insensitive to changes in the osmolarity of the growth medium in contrast to the wild type cells which exhibit osmoregulation of OmpF and OmpC synthesis. All beta-lactam-resistant mutants examined are resistant to novobiocin.

Sixteen heat shock proteins (Hsps) have been identified in the hypertoxinogenic strain 569B of Vibrio cholerae which are synthesized in response to small and large elevations of temperature. The induction of the Hsps is necessary for the cells to survive the deleterious effects of heat. There is no difference in the pattern of induction of the Hsps in V. cholerae strains varying in levels of toxinogenicity. One of the major low-molecular-mass Hsps, a 16-kDa protein, is preferentially degraded following shift down of temperature. This protein is induced at a much lower level at high temperatures in cells maintained in the laboratory for a prolonged period. The only Hsp located in the outer membrane of V. cholerae cells is a 23-kDa protein. Western immunoblot analysis with human immune sera collected from convalescent cholera patients revealed that this protein is markedly immunogenic. The human immune serum also reacted with the 69- and 16-kDa major Hsps and the 88-, 66-, and 46-kDa Hsps but not with the 61-kDa major Hsp identified as the groEL gene product. All major Hsps reacted with rabbit anti-V. cholerae sera. Ethanol stress leads to the induction of four of the major Hsps and three additional proteins.

The DNA adenine methyltransferase (MTase)-encoding gene (dam) of Vibrio cholerae, an organism belonging to the family Vibrionaceae, has been cloned and the complete nucleotide (nt) sequence determined. V. cholerae dam encodes a 21.5-kDa protein and is directly involved in methyl-directed DNA mismatch repair. It can substitute for the Escherichia coli enzyme and can suppress the phenotypic traits associated with E. coli dam mutants. Overproduction of V. cholerae Dam MTase does not result in hypermutability in either V. cholerae or E. coli cells. Overproduction of V. cholerae Dam in a pUC plasmid, however, fails to suppress the 2-aminopurine (2-AP)-sensitive phenotype of E. coli dam mutants. Homology between the nt and deduced amino acid (aa) sequences of the E. coli and V. cholerae dam genes is only 30-35%.

A clinical isolate of Vibrio cholerae 01 was identified which did not possess the heat-labile (CT), the heat-stable (ST) or the zonula occludens (Zot) toxin genes. Rabbit ileal loop assays showed that no other CT-like toxin was produced by this strain. The partly deleted cholera toxin gene which carries the intact gene for the B subunit was cloned and the recombinant plasmid, pURD110, was introduced into this non-toxinogenic natural human isolate. The transformed cells (strain URD2) secreted the B subunit gene product which competed with the holotoxin secreted by the hypertoxinogenic strain 569B of V. cholerae for the GM1 ganglioside binding sites in vivo. This strain can colonize the rabbit intestine as detected by the removable intestinal tie adult rabbit diarrhoea (RITARD) model. This construct has an advantage over other live oral attenuated V. cholerae strains used as vaccines in that the latter strains were made non-toxinogenic by only deleting part of the gene coding for the A subunit of cholera toxin while the strain described here is naturally non-toxinogenic.

The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The NotI and SfiI digestion patterns of the genomes of several V. cholerae strains belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae.

A new choleraphage, psi 92, with an icosahedral head and no tail structure has been described. This phage has a wide host range. It infects and lyses strains belonging to different serovars and biotypes of Vibrio cholerae. The phage utilises host DNA degradation products for its own DNA synthesis. psi 92 contains a linear double-stranded DNA of 56 kbp. A physical map of the phage genome has been constructed using the restriction enzyme KpnI. Intracellular development of phage psi 92 following infection was examined by thin-section electron microscopy.

The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations.

Choleraphage phi 149 differentiates the two biotypes, classical and el tor, of Vibrio cholerae. This phage cannot replicate in V. cholerae biotype el tor cells because the concatemeric DNA intermediates produced are unstable and cannot be chased to mature phage DNA. A V. cholerae biotype el tor gene coding for a 14,000-Da inner membrane protein which destabilizes the concatemeric DNA intermediates by hindering their binding to the cell membrane has been identified. Presumably, a 22,000-Da V. cholerae biotype el tor protein might also have a role in conferring phage phi 149 resistance to cells belonging to the biotype el tor. A nucleotide sequence homologous to the 1.2-kb V. cholerae biotype el tor DNA coding for both the 14,000- and 22,000-Da proteins is present in all strains of classical vibrios but is not transcribed. The nucleotide sequence of the gene coding for the 14,000-Da protein has been determined.

Unlike other gram-negative enteric bacteria, Vibrio cholerae cells were equally susceptible to penicillin and ampicillin and in general more susceptible than Escherichia coli to most of the beta-lactam antibiotics. The turbidity of penicillin-treated cultures contained to increase exponentially for about 3 h, although the cell viability declined rapidly within 30 min of penicillin addition. Prolonged treatment with beta-lactam antibiotics produced cells resistant to these antibiotics. A fluctuation test indicated that this resistance might be due to adaptive mutation. Cells resistant to a beta-lactam exhibited broad cross-resistance to other beta-lactam antibiotics. A new 12,000-Da outer membrane protein was detected both in beta-lactam-resistant cells and in wild-type cells growing in medium containing beta-lactam antibiotics. While the penicillin-resistant cells had all of the penicillin-binding proteins (PBPs) present in the parental cells, significant differences in the relative proportion of low-molecular-weight PBPs were seen. The low-molecular-weight PBPs from resistant cells seemed to form more stable complexes with penicillin than those from the parental strain.

Vibrio cholerae 569B was found to be highly sensitive to a wide range of chemicals, particularly hydrophobic compounds and neutral and anionic detergents. The phospholipid profile of the outer membrane was similar to that reported for other Gram-negative bacteria. The lipopolysaccharide (LPS) contained O-antigenic sugars and exhibited heterogeneity. In addition, the LPS moiety was characterized by a relatively low negative charge. Analysis by topological probes revealed the presence of a significant amount of exposed phospholipids in the outer membrane. The reduced negative charge of LPS molecules and the exposed phospholipids present in the outer membrane could be important in the increased permeation of exogenous compounds in V. cholerae.

The lack of an efficient transformation system in Vibrio cholerae was a handicap in the genetic manipulation of this important human pathogen. Since V. cholerae cells secrete DNases, this may interfere with the uptake of DNA. The present report describes the approaches taken for transforming V. cholerae cells with plasmid DNA, by overcoming this DNase barrier. The partial success of transforming DNase-negative mutants confirmed the role of DNase in the nontransformability of the wild-type cells. Successful transformation was carried out following removal of DNases from the periplasmic space. This was achieved by treating the cells with Mg2+ and Ca2+ ions to allow the DNase to be released, and then holding them under conditions where the remaining DNase activity was minimized before adding DNA to the competent cells. Transformation efficiencies of the order of 10(-5) per recipient cell were observed.

Eleven penicillin binding proteins (PBPs) of Vibrio cholerae have been identified using [125I] labelled p-hydroxybenzyl penicillin (PenX). These proteins are localised in the inner membrane and have molecular weights ranging from 97,000 to 22,000. Neutral hydroxylamine released the labelled PenX from the PBPs and pretreatment with cold benzyl penicillin inhibited labelling completely. The PBP 4 is the most sensitive target for cephaloridine and aztreonam. Cephaloridine also binds to three other high molecular weight PBPs, 1, 2 and 3. Aztreonam, in addition to PBP 4, has affinity for another low molecular weight PBP, PBP 7. Mecillinam has affinity for PBPs 1, 4 and 11.

Vibrio cholerae mutants resistant to common antibiotics and neutral and anionic detergents were isolated. Analysis of isolated outer membranes revealed a significant deficiency in the acylation of lipid A in the resistant strains. The content of amide-linked and ester-bound fatty acids in the lipid A of the mutant strains compared to that of the wild type was about 50-56% and 29-37% respectively. This defect was specific for lipid A as there was no change in the acylation of phospholipids. The reduction in fatty acid content of lipid A was reflected in the altered endotoxic properties in the mutant strains.

Cells of Vibrio cholerae underwent rapid autolysis when suspended in media of low osmolarity under non-growing conditions. Chaotropes like urea and guanidine. HCl which are potent protein denaturants caused complete and immediate lysis of whole cells. This unique sensitivity of V. cholerae to protein denaturants led to the development of a rapid method for the selective isolation of the outer membrane upon treatment of whole cells with urea. The composition of the outer membrane isolated from both whole cells and crude envelopes by treatment with urea was comparable with that of the outer membrane isolated by other conventional methods.

The synthesis of alkaline phosphatase by two strains of Vibrio cholerae belonging to the Inaba and Ogawa serotypes has been examined in relation to the phosphate concentration of the culture medium. The synthesis of the enzyme in both strains was repressed in cells grown in the presence of a high concentration of inorganic phosphate. Lowering the phosphate content of the growth medium led to a derepression of enzyme activity. The presence of glucose in low phosphate medium stimulated the degree of derepression. The synthesis of the enzyme by strain Inaba 569B was more sensitive to inorganic phosphate than that of strain Ogawa 154. The enzyme was presumably located in the periplasmic space since it was released when the organisms were converted to spheroplasts.

A library containing more than 80% of the Vibrio cholerae genome was constructed by cloning BamH1 restriction fragments into pBR322. Using interspecific complementation of an Escherichia coli recA mutant with plasmids containing the gene bank of V. cholerae, a recA-like gene was identified. The recombinant plasmid, designated as pDP145, contained a 1.45 kb segment of V. cholerae DNA which codes for a protein of molecular weight 39,000. The product of this gene confers methyl methane sulphonate resistance on the E. coli recA mutant, suppresses its ultraviolet (UV) light sensitive phenotype and has proteolytic activity on the phage lambda repressor. Induction of a 39,000 dalton protein in UV-irradiated V. cholerae cells was demonstrated.

The mutL and mutS genes of Vibrio cholerae have been identified using interspecific complementation of Escherichia coli mutL and mutS mutants with plasmids containing the gene bank of V. cholerae. The recombinant plasmid pJT470, containing a 4.7 kb fragment of V. cholerae DNA codes for a protein of molecular weight 92,000. The product of this gene reduces the spontaneous mutation frequency of the E. coli mutS mutant. The plasmid, designated pJT250, containing a 2.5 kb DNA fragment of V. cholerae and coding for a protein of molecular weight 62,000, complements the mutL gene function of E. coli mutL mutants. These gene products are involved in the repair of mismatches in DNA. The complete nucleotide sequence of mutL gene of V. cholerae has been determined.

Intracellular development of choleraphage phi 149 following infection of Vibrio cholerae and Vibrio eltor cells under different conditions was examined by thin-section electron microscopy. Degradation of the host DNA following infection and formation of mature phage particles inside the infected cells were demonstrated. The concatemeric DNA intermediate formed during intracellular replication of phi 149 DNA in permissive hosts was resolved. In confirmation of biochemical evidence, no concatemeric DNA intermediate was observed for infection in high-phosphate medium.

Choleraphage phi 138 contains a linear, double-stranded, circularly permuted DNA molecule of 30 X 10(6) daltons or 45 kilobase pairs. Upon infection, the host DNA is degraded, and synthesis of phage-specific DNA is detectable 20 min after infection. The phage utilizes primarily the host DNA degradation products for its own DNA synthesis. A physical map of phi 138 DNA was constructed with the restriction endonucleases Bg/II, HindIII, and PstI. A concatemeric replicative DNA intermediate equivalent to eight mature genome lengths was identified. The concatemer was shown to be the precursor for the synthesis of mature bacteriophage DNA which is subsequently packaged by a headful mechanism.

DNA repair-proficient and -deficient strains of Vibrio cholerae were used to examine host cell reactivation, Weigle reactivation and photoreactivation of u.v.-irradiated cholera bacteriophages. U.v. light-induced DNA damage in phages of different morphological and serological groups could be efficiently photoreactivated. Host cell reactivation of irradiated phages of different groups was different on the same indicator host. Phage phi 149 was the most sensitive, and phi 138 the most resistant to u.v. irradiation. While phi 138 showed appreciable host cell reactivation, this was minimal for phi 149. Attempts to demonstrate Weigle reactivation of u.v.-irradiated cholera phages were not successful, although u.v.-induced filamentation of host cells was observed.

The rate and extent of lysis of Vibrio cholerae cells under nongrowing conditions were dependent on the osmolarity of the growth medium. Gross alterations in cellular morphology were observed when V. cholerae cells were grown in media of high and low osmolarity. The rate of lysis of V. cholerae cells under nongrowing conditions increased after treatment with chloramphenicol. Chloramphenicol-treated V. cholerae 569B cells showed formation of sphaeroplast-like bodies in medium of high osmolarity, but not in low osmolarity. Changes in the osmolarity of the growth medium also regulated the expression of the outer membrane proteins. This regulation was abolished if V. cholerae cells were grown in Pi-depleted medium. Analysis of the lytic behavior and composition of outer membrane proteins of an osmotically fragile mutant strain revealed a similar dependence on the osmolarity of the growth medium.

Repair of ultraviolet-light-induced DNA damage in a highly pathogenic Gram-negative bacterium, Vibrio cholerae, has been examined. All three strains of V. cholerae belonging to two serotypes, Inaba and Ogawa, are very sensitive to ultraviolet irradiation, having inactivation cross-sections ranging from 0.18 to 0.24 m2/J. Although these cells are proficient in repairing the DNA damage by a photoreactivation mechanism, they do not possess efficient dark repair systems. The mild toxinogenic strain 154 of classical Vibrios presumably lacks any excision repair mechanism and studies of irradiated cell DNA indicate that the ultraviolet-induced pyrimidine dimers may not be excised. Ultraviolet-irradiated cells after saturation of dark repair can be further photoreactivated.

Vibrio cholerae mutants sensitive to 2-aminopurine (2AP) but with DNA adenine methylase activity similar to parental cells have been isolated. The mutant strains were sensitive to ultraviolet light (UV), methyl methane sulphonate (MMS) and 9-aminoacridine. The spontaneous mutation frequency of the mutants were not significantly affected. Attempts to isolate dam V. cholerae cells by screening 2AP sensitive cells have not been successful. All the mutant phenotypes could be suppressed by introducing the plasmid pRB103 carrying the dam gene of Escherichia coli into the mutant cells.

A radiation-sensitive mutant of the hypertoxinogenic strain 569B of Vibrio cholerae was isolated and characterized. The mutant, designated V. cholerae 569Bs, lacks both excision- and medium-dependent dark-repair mechanisms of UV-induced DNA damage while retaining the wild-type photoreactivating capability. Analysis of the UV-irradiated cell DNA by velocity sedimentation in alkaline sucrose gradient suggests that UV-induced pyrimidine dimers may not be incised in these cells. In contrast to the wild-type cells, the mutant cell DNA was degraded after treatment with nalidixic acid. The mutant cells failed to produce any detectable amount of cholera toxin as measured by ileal-loop assay.

Alkaline phosphatase has been purified to homogeneity from two strains of Vibrio cholerae. The enzymes from both strains are single polypeptides of molecular weight 60,000. Both of the enzymes have pH optima around 8.0 and can act on a variety of organic phosphate esters, glucose-1-phosphate being the best substrate. The enzymes are unable to hydrolyze ATP and AMP. Although they have identical Km values, the two enzymes differ significantly in Vmax with p-nitrophenyl phosphate as substrate. The enzymes from the two strains also differ in their sensitivity to EDTA, Pi, and metal ions and activities of the apoenzymes. Ca2+ reactivated the apoenzymes most.

Attempts to identify an umuDC analog, using interspecific complementation of Escherichia coli mutants with plasmids containing a gene bank of Vibrio cholerae, were not successful. The DNA from none of the vibrio species examined including marine vibrios hybridized to E. coli umuC and umuD gene sequences. These cells are not mutable by ultraviolet (UV) light and cannot Weigle-reactivate UV-irradiated choleraphages, suggesting that vibrios are deficient in the umuDC operon. This possibility is supported by the fact that when the plasmid pKM101 carrying the mucAB genes is introduced into V. cholerae cells, they acquire the UV-mutable phenotype and UV-irradiated choleraphages can be Weigle-reactivated.